Supplementary MaterialsSupplemental Material. severe dose-limiting toxicities (1, 2). Efficacy of cytokine-based therapies is limited by an inability to deliver them to the proper location and an incomplete knowledge of the effects of particular cytokines in various cancer types. The construction of antibody-cytokine fusions is an established preclinical approach to target cytokine therapy to the tumor microenvironment. However the size of these adducts results in persistence in circulation and comparatively poor tissue penetration. As an alternative Pifithrin-alpha inhibition to full-sized antibodies as fusion partners, we developed alpaca-derived heavy chain-only antibody fragments (VHHs) against programmed death-ligand 1 (PD-L1)(3). In cancer patients, PD-L1 expression is confined largely to the tumor microenvironment, being expressed on a wide variety of tumor cell types and tumor infiltrating myeloid cells (4). VHHs, at ~15 kDa, more readily penetrate tumors than full-sized antibody fusions. Antibody-cytokine fusions designed for delivery of IL2 to the tumor microenvironment have been tried before (5). Unfortunately, the high affinity of IL2 for its receptors and the abundant expression of IL2 receptors in peripheral blood, spleen, and liver, leads to a distribution of the antibody-cytokine fusion that is dictated primarily by the cytokine partner rather than the antibody to which it is attached for targeted delivery. Mathematical modeling predicts that smaller sized protein fusions would allow IL2 to concentrate in the tumor microenvironment (5). Although VHHs have a short circulatory half-life of ~30 minutes, they can remain bound to their targets for more than 24 hours(6, 7). The rapid clearance of VHHs from the circulation, combined with their high affinities and long tissue half-lives, means that it should be possible to concentrate VHHs and attached payloads in the tumor microenvironment while minimizing systemic exposure. VHHs accept a variety of payloads, including radioisotopes and cytokines, which can be installed in a straightforward manner(3, 8-11). Disruption of immune checkpoint interactions by monoclonal antibodies (mAbs) has replaced chemotherapy as the standard of care for metastatic melanoma and is similarly promising in the treatment of other cancers (4, 12-14). Checkpoint blockade largely augments a pre-existing T-cell response, and Pifithrin-alpha inhibition has had far less efficacy against tumors that are poorly infiltrated by T cells at baseline (15, 16). Cytokine-based therapies may be particularly promising as a means of manipulating the immune microenvironment. We have chosen to focus on pancreatic ductal adenocarcinoma (PDAC), a tumor type unresponsive to checkpoint blockade (13, 17, 18). Pancreatic ductal adenocarcinoma is one of the deadliest cancers, with a 5-year survival rate of 8% (19). The disease is rapidly metastatic, and the majority of primary pancreatic tumors are inoperable due to invasion of the surrounding vasculature. Pancreatic tumors are dense, fibrotic masses that preclude adequate drug delivery and may limit accessibility for full-sized antibodies (20). The dense stroma creates a nutrient-poor, immunosuppressive environment. Pifithrin-alpha inhibition Approximately 60% of human PDAC tumors express PD-L1 (staining 10% by immunohistochemistry) (21). The majority of immune cells, both in human tumors and mouse models, are cells of the myeloid lineage, with both granulocytic and monocytic myeloid-derived suppressor cells (MDSCs), as well as tumor-associated macrophages (TAMs) contributing to local immunosuppression. Many human and mouse PDAC tumors are devoid of CD8+ Pifithrin-alpha inhibition T-cell infiltrates at baseline, suggesting that T cells are either not primed against PDAC antigens, fail to reach the tumor at all or are Rabbit polyclonal to ESR1.Estrogen receptors (ER) are members of the steroid/thyroid hormone receptor superfamily ofligand-activated transcription factors. Estrogen receptors, including ER and ER, contain DNAbinding and ligand binding domains and are critically involved in regulating the normal function ofreproductive tissues. They are located in the nucleus , though some estrogen receptors associatewith the cell surface membrane and can be rapidly activated by exposure of cells to estrogen. ERand ER have been shown to be differentially activated by various ligands. Receptor-ligandinteractions trigger a cascade of events, including dissociation from heat shock proteins, receptordimerization, phosphorylation and the association of the hormone activated receptor with specificregulatory elements in target genes. Evidence suggests that ER and ER may be regulated bydistinct mechanisms even though they share many functional characteristics rendered non-functional due to early establishment of an immunosuppressive microenvironment (22). Strategies to reduce infiltration of myeloid Pifithrin-alpha inhibition cells or to reprogram these cells to an alternative fate can enhance CD8+ T-cell infiltration and synergize with checkpoint blockade therapy (23-25). Reprogramming of myeloid cells can lead to.